Cellular proliferation and differentiation events are tightly controlled during normal development and miss regulation of these events leads to abnormal development and disease, such as cancer. Therefore, understanding the precise complements of gene functions regulating cellular proliferation and differentiation will not only provide insights of normal development, but also provide clues to understand the etiology of tumorigenesis. My previous studies on Six6, a member of mammalian sine oculis family, revealed that it controls retinal and pituitary precursor cell proliferation by directly regulating key components of cell cycle machinery, such as cyclin-dependent protein kinase inhibitors. Functional importance of Six-family genes in tumorigenesis is further suggested by the observations that Six1 is selectively up regulated at the late stage of G1 cell cycle and promotes G2/S cell cycle transition. Also, Six1 is found to be aberrantly expressed in more than 90% of human metastatic breast cancers as well as in Wilm's tumor. Together, these data suggest that Six-family factors are key components in regulating cellular proliferation event. Further, preliminary results demonstrated that Six1 is highly expressed in pituitary gland and kidney, suggesting possible functional importance of Six-genes in organ specification, such as neural endocrine and kidney system. Indeed, our initial analyses of Six1 mutant mice demonstrated the complete absence of kidney formation. In order to fully understand the underlying molecular mechanisms of Six-gene functions and in turn to provide clues to prevent their aberrant functions in kidney disease, such as Wilm's tumor, I propose: 1) To define the roles of Six1 during kidney formation; and 2) To identify Six1 cofactors that modulate its activities and to identify potential Six1 target genes. Both genetic loss-of-function and gain-of-function approaches will be used to elucidate the functional importance of Six-family genes and the underlying mechanisms that regulate their functions. Chromatin immunoprecipitation-coupled promoter microarray and Affymetix(r) GeneChips bioinformatics approaches will be used to identify Six1 direct and indirect target genes. To begin to understand the functional switching of Six1 activity, the tandem affinity purification method will be used to biochemically purify specific Six1-complex followed by mass spectrometry protein identification.